Expression of TNF- α at mRNA level in Thp-1 cells exposed to LPS and HSP60: Possible impact on diabetic vascular inflammation

Diabetes mellitus is a chronic disease characterized by hyperglycaemia. It occurs when the b cells of the pancreas produce either low levels of insulin or no insulin at all. The chronic hyperglycaemic condition of diabetes is associated with the dysfunction, long-term damage and failure of organs such as the heart and blood vessels. Diabetes is closely linked to the prevalence of cardiovascular diseases, which is one of the highest causes of death worldwide. In particular, the hyperglycaemic and chronic inflammatory state of diabetes has been linked with atherogenesis, which is when fatty deposits build up in the arteries and they become inflamed. It still remains unknown how diabetes contributes to atherosclerosis. Heat Shock Protein 60 (HSP60) is an important endogenous inflammatory mediator elevated in diabetic patients. HSP60 can be released from various cell types, including monocyte cells, which are the most common cell type found in atherosclerotic plaques. The mechanism behind how HSP60 triggers endothelial cell inflammation remains unknown, but has been hypothesised to be similar to that of a Toll like receptor 4 ligand, lipopolysaccharide (LPS). Understanding the molecular mechanism of HSP60 could give insights into the pathological, cellular and molecular basis of vascular inflammation in diabetes as well as other vascular diseases such as cardiovascular disease. It is hypothesized that monocyte-derived HSP60 has a role in causing inflammation in the diabetic vasculature through the expression of pro- inflammatory cytokines such as TNF-α, which could contribute to atherosclerosis. In this study a human monocyte cell line (Thp-1) was used to mimic monocyte behaviour. First a growth curve was established, which indicated when the cells were in their exponential phase. This enabled RNA to be extracted during the period when cells were growing the fastest. Thp-1 cells were then exposed to LPS for 6, 12, and 24 hours to determine the expression of TNF-α. After conducting RT-PCR, a 12 hour time point was selected. Since inconsistent results were obtained using this semi- quantitative analysis, qPCR was carried out for the rest of the project. Thp-1 cells were then exposed to 50ng/mL and 100ng/mL of LPS for 12 hours. After conducting and analysing qPCR using the delta-delta Ct method and the Pfaffl method, a ~2 fold increase was seen in the expression of a pro-inflammatory cytokine, TNF-α. These results however were not significant (determined by a Student’s T test). In order to obtain a higher level of TNF-α expression, this experiment was repeated with 10ng/mL and 50ng/mL of LPS for 4 hours, based on previous published research. These results were analysed using the delta-delta Ct method and the Pfaffl method. The results were not what was expected; only an insignificant